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La diagnosi chimico-tossicologica di esposizione a sostanze d'abuso mediante analisi dei capelli
No full textNell'esigenza di superare i limiti temporali imposti dai liquidi biologici tradizionali (sangue e urina), l`analisi dei
capelli si è affermata come valido strumento per accertamenti dell`uso non recente di sostanze stupefacenti e
psicotrope. I capelli, grazie alla loro capacità di sequestro, consentono infatti un significativo ampliamento della
finestra temporale di rilievo delle sostanze assunte (settimane/mesi a fronte di ore e/o giorni riferibili a sangue e
urine), rappresentando così un utile complemento alle matrici tradizionali. Inoltre, attraverso l’analisi
segmentale, essi permettono di ricavare informazioni sulla storia del consumo. Pur a fronte dei numerosi
vantaggi di ordine metodologico e pratico (facile prelievo e conservazione), numerosi sono i problemi ancora
aperti nell'analisi dei capelli. Tra questi sono da annoverare, ad esempio, la possibile rimozione delle sostanze
incorporate ovvero l'interferenza nell'analisi in seguito a trattamenti cosmetici aggressivi, la contaminazione
passiva, la forte dipendenza del risultato dal metodo analitico impiegato e, soprattutto, dal metodo di
preparazione del campione. Obiettivo di questa tesi è stato quello di esaminare alcune di queste criticità e, in
particolare: (a) la rimozione di alcune droghe d’abuso - oppiacei e cocaina (CO) – oltre che di un marcatore di
abuso alcolico, l’etilglucuronide (EtG), dai capelli in seguito ad adulterazione riferibile a due tipi di trattamenti,
vale a dire l’utilizzo di lozioni “anticaduta” contenenti Minoxidil e l’impiego di una lozione decolorante
aggressiva; (b) lo sviluppo e la validazione di un metodo specifico per la determinazione quantitativa di analiti
impegnativi quali la buprenorfina (BPR) e il suo metabolita nor-buprenorfina (norBPR) e (c) l’individuazione,
attraverso l’elaborazione della casistica ultradecennale delle analisi dei capelli richieste al Laboratorio di Analisi
Chimico-Tossicologiche dell’Università di Pavia da parte delle Commissioni Mediche Locali (CML) per le
patenti, di una strategia diagnostico-interpretativa efficace ed efficiente, anche in riferimento all’analisi
segmentale dei capelli.
Gli studi sperimentali realizzati nell'ambito della tesi hanno permesso di accertare che: le lozioni “anticaduta”
contenenti minoxidil interferiscono con l’analisi dei capelli impedendo l’identificazione della cocaina e dei suoi
metaboliti, anche se il problema è in parte ovviabile attraverso la messa in atto di alcuni accorgimenti analitici
(omissione della fase di derivatizzazione); la decolorazione è in grado di rimuovere quantitativamente sia gli
oppiacei, sia l'EtG dai capelli, mentre nel caso della cocaina e metaboliti la rimozione non è generalmente
quantitativa; BPR e norBPR sono rilevabili nei capelli di soggetti trattati con BPR (2-8 mg/die) anche se in
concentrazioni contenute (tra 0.01 e 0.1 ng/mg nel caso della BPR) e tali da richiedere l'impiego di un metodo
caratterizzato da elevata sensibilità analitica; l'elaborazione della casistica delle analisi effettuate nel periodo
1996-2007 ha evidenziato il crescente ricorso all'analisi dei capelli da parte delle CML (quadruplicazione delle
richieste annuali nel periodo esaminato), la tendenza all'aumento delle positività per cocaina a fronte della
riduzione di quelle per oppiacei e la dipendenza dell'esito (positivo/negativo) dell'analisi dalla richiesta di analisi
segmentale (analisi separata dei segmenti prossimale e distale di 3 cm ciascuno, come previsto dalle linee guida
della Regione Lombardia) piuttosto che di analisi in toto (segmento unico di 6 cm), probabilmente a causa del
possibile effetto di diluizione nel secondo caso.Owing to the necessity to overcome the time limits of conventional biological substrates (blood and
urine), hair analysis is increasingly being used as an efficient analytical tool to ascertain remote and
habitual exposure to drugs of abuse. As a matter of fact, hair analysis allows a significant widening
of the detection time-window (weeks/months instead of hours/days in the case of blood and urine),
thus representing a useful complementary diagnostic tool to the analysis of conventional matrices.
Moreover, through segmental analysis, it is possible to get information even on the history of use of
a substance. Despite the several methodological as well as practical advantages (easy collection and
storage) a number of issues on hair analysis still remain open. Among these, the possibility of
removal of incorporated substances after strong cosmetic treatments, passive contamination, the
strong method-dependence of analytical results and, most of all, the dependence of results on the
sample preparation procedure, should be counted. The aim of this thesis was to tackle some of these
issues and, in particular: (a) the removal of drugs of abuse - opiates and cocaine – and of a marker
of chronic alcohol abuse, ethyl glucuronide (EtG), from hair as a consequence of two different
treatments (with an anti hair loss solution containing Minoxidil and with a strong bleaching lotion);
(b) the development and validation of a specific method for the quantification of buprenorphine
(BPR) and its metabolite nor-buprenorphine (norBPR) and (c) the definition, through the
elaboration of data on hair analysis for driving license renewal/re-issue collected during over 10
years by the Laboratory of Chemical-Toxicological Analysis of the University of Pavia, of an
efficient diagnostic-interpretative strategy, with specific reference to the segmental hair analysis.
The experimental studies carried out during this thesis permitted to ascertain that: the anti hair loss
lotions, containing minoxidil, interfere with hair analysis thus preventing the identification of
cocaine and its metabolites, although the problem can be partially solved by adopting a
modification in the analytical procedure (omission of the derivatization step); hair bleaching
quantitatively removes both opiates and EtG from the hair matrix, whereas for cocaine and
metabolites the removal is generally not quantitative; BPR and norBPR are detectable in subjects
under BPR treatment (2-8 mg/day), although the concentrations found are low (from 0.01 to 0.1
ng/mg for BPR), thus requiring a highly sensitive analytical method for their detection; the
elaboration of data collected in the period 1996-2007 highlighted an increasing demand of hair
analyses (quadruplication of the number of requests per year), an increasing trend of cocainepositive
cases accompanied by a decreasing trend in opiate-positive cases, and the dependence of
results (positive/negative) on the type of request: either segmental hair analysis (analysis of the two
3-cm proximal and distal segments, as suggested in the Guidelines on hair analysis for drugs of
abuse issued by Lombardy) rather than the analysis of the whole 6-cm segment, likely due to a
dilution effect in the latter case
Ethyl glucuronide and ethyl sulphate determination in serum by liquid chromatography-electrospray tandem mass spectrometry
No full textBackground: Ethyl glucuronide (EtG) and ethyl sulphate (EtS) are two ethanol metabolites that can be detected in serum up to 8 It after ethanol elimination. Their presence is therefore indicative of recent ethanol consumption in case of delayed sampling after an event (e.g. car crash).
Methods: A LC-ESI-MS-MS method for the determination of EtG and EtS in serum was developed and validated. Two product ions together with the parent ions were monitored for identification. Pentadeuterated-EtG was used as internal standard.
Results: Excellent linearity for EtG (from 0.22 to 45 mu mol/l) and EtS (from 0.40 to 80 mu mol/1) was observed (r(2) >= 0.9998). LOD and LLOQ were 0.04 and 0.20 mu mol/l for EtG and 0.08 and 0.40 mu mol/l for EtS, respectively. Accuracy (bias) and precision (relative standard deviation), studied at four different quality control levels, were always better than 7%. Matrix effects were found to be negligible. The method was applied to several samples obtained from known alcoholics and social drinkers.
Conclusions: A sensitive and specific determination of EtG and EtS in serum samples was achieved despite a simple and fast sample preparation. To our knowledge, this is the first fully validated method for the simultaneous determination of the two alcohol metabolites in seru
Hair analysis in workplace drug testing and driving licence regranting as a tool to disclose patterns of drug use
No full textBackground: In Italy, hair analysis is used in the second level-tests of workplace drug testing (WDT) protocol and for driving licence regranting/renewal (DLR). The WDT National Protocol includes two-level tests. The second level of the protocol is required when in the first level-tests urine sample results positive. In this case, the worker is obliged to go to the Public Drug Treatment Unit to undergo an eventual diagnosis of drug dependence. The second level-tests consist of analysing both urine and hair for opiates, cocaine, amphetamines, cannabinoids, methadone and buprenorphine.
As far as driving licence regranting/renewal is concerned, subjects are instead always obliged to undergo toxicological analyses if they have either a documented past or a suspect present history of drug abuse, ( art. 187 “Driving under influence of drugs”), or if they are forced by the prefecture or by the traffic control authority. In some Regions such as Lombardy, hair analysis is used to rule out illicit drug use.
This study presents the data obtained from hair analysis performed in the Laboratory of Forensic Toxicology of the University of Pavia in the last two years.
Method: A total of 1017 hair samples were analysed for WDT (322) and DLR (695). Analyses were carried out by GC-MS using the method routinely applied on hair samples.
Results and Discussion: The positive rate for the WDT second leve-test was 45% versus the 10% for the DLR. The different percentage of positive results demonstrate how hair analysis is useful both for the diagnosis of drug dependence and as deterrent of drug consumption. As regard to positive results distribution, a cocaine abuse emerged in 74.6% of workers, while drivers resulted positive mainly for cannabinoids (41%), and cocaine (39%). On the contrary, the second main consumption in WDT analysis resulted the polydrug abuse (16,5%). Opiates percentage was 1.4% in both cases while positive samples for methadone were 9 in the DLR analysis versus 3 in the WDT one. MDMA was present only in two workers and in association with cocaine
Effect of bleaching on ethyl glucuronide in hair: An in vitro experiment
No full textINTRODUCTION: Ethyl glucuronide in hair (HEtG) has recently gained great attention, because of its high sensitivity and specificity in the diagnosis of chronic alcohol abuse. Due to its high polarity hydrophilicity, a strong hair treatment followed by a shampooing may lead to removal/degradation of this molecule from hair matrix.
AIM: To set up an in vitro study in order to evaluate the ability of bleaching of modifying HEtG test results.
METHODS: Thirty hair samples from teetotalers (n=5), social drinkers (n=4) and heavy drinkers (n=21), after an informed written consent, were collected and divided longitudinally into four aliquots. The first aliquot was kept untreated and was processed following the method routinely used in our lab for the determination of HEtG (double washing with methanol/dichloromethane, overnight incubation in water, and LC-MS/MS analysis, LLOQ: 3pg/mg). To the other three aliquots a commercially available bleaching solution was applied, according to the manufacturer's instructions. One out of the three aliquots was submitted to the analysis by following the same procedure used for the untreated sample. The other two were submitted to a purification step before LC-MS/MS analysis, by using two different SPE cartridges (aminopropyl and dimethyl butylamine).
RESULTS: HEtG levels in the untreated samples from social drinkers and heavy drinkers ranged from 7.7 to 149.0pg/mg. All the samples from teetotalers tested negative. The treated samples processed without any SPE extraction and with aminopropyl cartridges showed a relevant ion suppression for both EtG and D(5)-EtG (IS) signals. Samples treated with the bleaching solution and extracted with dimethyl butylamine cartridge allowed to sensitively reduce ion suppression (less than 35%) and to verify that EtG, after a strong treatment like bleaching, completely disappears.
CONCLUSIONS: This in vitro study showed that HEtG disappears from hair matrix after a strong hair treatment. It is not clear whether the mechanism involved is chemical degradation or physical removal from the damaged keratinic matrix. However, owing to the highly hydrophilic character of the compound, the second mechanism seems more likely to occur. Finally, bleaching solutions could lead to a heavy ion suppression of this metabolite that may be avoided by using an SPE purification before instrumental analysis
Markers of chronic alcohol use in hair: Comparison of ethyl glucuronide and cocaethylene in cocaine users
No full textTwo direct ethanol metabolites, namely ethyl glucuronide (EtG) and cocaethylene (CE), in the hair of cocaine (COC) users were compared in this study.
Hair samples (n = 68) were submitted to the determination of EtG (by liquid chromatography-electrospray-tandem mass spectrometry) and of COC and metabolites, including CE (by gas chromatography-mass spectrometry). Quantitative and qualitative results were compared.
No quantitative correlation was found between EtG and CE, as well as between EtG and the cocaethylene concentration divided by the concentration of COC and its metabolites (benzoylecgonine and ecgonine methylester, as COC equivalents). Nevertheless, many factors are supposed to affect the amount of the two substances incorporated in the hair matrix, such as the subject's habits in ethanol and COC use, genetic variability in the metabolism of both substances, and the different chemical and physical properties of EtG and CE.
When establishing a cut-off of 4 pg/mg for EtG and of 200 pg/mg for CE, 47 samples tested positive for EtG and 41 samples tested positive for CE; 12 samples out of the 47 EtG-positives tested negative for CE (25%), whereas 6 samples out of the 41 CE-positives tested negative for EtG (15%). According to these data, EtG appears to be a more sensitive and specific marker of non-moderate alcohol users than C
Treatments against hair loss may hinder cocaine and metabolites detection
No full text: Recently, some of the hair samples that we routinely analyse for drugs of abuse did not produce valid results for cocaine and metabolites. A series of very intense interfering peaks with ion fragments common to cocaine (CO), and benzoylecgonine (BE) were found to cover up the "cocaine" region of the chromatogram. In one of these cases the subject declared he had used a lotion containing Minoxidil in order to prevent hair loss. Starting from this observation we found that the interfering peaks belonged to four different TMS derivatives of Minoxidil. Minoxidil interference was further investigated by applying Tricoxidil(®), a Minoxidil solution, to the hair of CO-free volunteers and to a CO-positive hair strand dipped into Tricoxidil. Hair were analysed before and after treatment. In both cases interfering peaks were absent in the chromatograms of untreated hair and appeared in treated hair. In the CO-positive hair detection of CO, BE and internal standard was completely hindered after treatment with Minoxidil. Attempts to separate interfering peaks from CO and metabolites by modifying the temperature programme failed. None of the hair washing methods tested (methanol; dichloromethane; sodium dodecyl sulphate water solution, 1% w/v followed by methanol; phosphate buffer 0.1 M, pH 6 followed by methanol) succeeded in removing Minoxidil interference. However, a simple solution to partially overcome the problem was to dry up the derivatised extract, reconstitute it in methanol (in order to switch back Minoxidil derivatives to the native molecule), and re-inject it: owing to the higher polarity, underivatised Minoxidil does not interfere any more with the chromatography of CO, at the expense of the disappearance of BE and ecgonine methyl ester both producing TMS derivatives. This strategy was applied to four real cases where Minoxidil interference was recognised: in two of these cases CO was detected. The problem of Minoxidil interference on CO detection may be limited to procedures involving trimethylsilylation, which is probably the most commonly adopted derivatisation in laboratories performing hair analysis for drugs of abuse
Treatments against hair loss may hinder cocaine and metabolites detection
No full textRecently, some of the hair samples that we routinely
analyse for drugs of abuse did not produce valid results
for cocaine and metabolites. A series of very intense interfering
peaks with ion fragments common to cocaine (CO),
and benzoylecgonine (BE) were found to cover up the
‘‘cocaine’’ region of the chromatogram. In one of these cases
the subject declared he had used a lotion containing
Minoxidil in order to prevent hair loss. Starting from this
observation we found that the interfering peaks belonged to
four different TMS derivatives of Minoxidil. Minoxidil
interference was further investigated by applying Tricoxidil
, a Minoxidil solution, to the hair of CO-free volunteers
and to a CO-positive hair strand dipped into Tricoxidil. Hair
were analysed before and after treatment. In both cases
interfering peaks were absent in the chromatograms of untreated
hair and appeared in treated hair. In the CO-positive
hair detection of CO, BE and internal standard was completely
hindered after treatment with Minoxidil. Attempts to
separate interfering peaks from CO and metabolites by
modifying the temperature programme failed. None of the
hair washing methods tested (methanol; dichloromethane;
sodium dodecyl sulphate water solution,1%w/v followed bymethanol; phosphate buffer 0.1 M, pH 6 followed by
methanol) succeeded in removing Minoxidil interference.
However, a simple solution to partially overcome the problem
was to dry up the derivatised extract, reconstitute it in
methanol (in order to switch back Minoxidil derivatives to
the native molecule), and re-inject it: owing to the higher
polarity, underivatised Minoxidil does not interfere any
more with the chromatography of CO, at the expense of the
disappearance of BE and ecgonine methyl ester both producing
TMS derivatives. This strategy was applied to four
real cases where Minoxidil interference was recognised: in
two of these cases CO was detected. The problem of
Minoxidil interference on CO detection may be limited to
procedures involving trimethylsilylation, which is probably
the most commonly adopted derivatisation in laboratories
performing hair analysis for drugs of abus
1996-2006: A decade of hair analysis for driving license applications. the experience of the department of legal medicine and public health of the university of pavia
No full textSegmental hair analysis in order to evaluate driving performance
No full textOn the 31st of July 2002 the Lombardy local government issued a memorandum, C.R. 35/SAN, providing "guidelines to investigate drugs of abuse addiction in order to judge driving performance".
About hair samples, this memorandum advises that the proximal lock of 6 cm-length would be analysed for opiates, cocaine, cannabinoids, amphetamine and derivatives, divided into two segments of 3 cm, each. The Local Medical Driving Licence Commissions (CML) can decide whether or not to enforce these instructions; from our survey it resulted that most CMLs do not abide by the memorandum, not requiring segmental analysis. The purpose of our study was to verify whether this procedural discordance could affect analytical results and, consequently, the evaluation of the subject's driving performance. We analysed hair samples taken from subjects who were requesting the renewal of their driving licence in our Laboratory during the period from 1 August 2002 to 31 December 2006. We divided samples into two groups: (1) samples previously analysed in one single segment which resulted positive for at least one analyte, but under the cut-off (0.5 ng/mg), were re-analysed in accordance with the guidelines; (2) samples previously processed following guidelines which resulted positive in one of the segments were newly analysed in a single segment. Comparing the new results with the original ones, an increase of positive results emerged in the first group. The second set of results fully supported the first ones.
These results underscore the importance of the 35/SAN memorandum, so if the guidelines had been followed there would have been a larger amount of driving licence renewal denie
Determination of buprenorphine and norbuprenorphine in hair by GC-MS
No full textBuprenorphine is a synthetic derivative of thebaine used as a substitute of heroin in detoxification programs. Buprenorphine was introduced for this application in May 2000 in Italy and its use for this therapeutic purpose has increased by over than 30% in three years. The aim of this study was to extend the method routinely applied in our laboratory to detect heroin metabolites, cocaine and amphetamines also to buprenorphine (BPR) and its metabolite norbuprenorphine (norBPR) in order to monitor BPR administration in detoxification programs as well as to detect drug abuse during detoxification. The analytical procedure was as follows: after a washing-step with methanol, hair was finely cut and incubated in HCl 0.1N (45°C, overnight). Nalorphine was chosen as internal standard. Purification of analytes was executed by solid phase extraction (Bond Elut Certify) which provided recoveries higher than 80% for both BPR and norBPR; purified extract was derivatised with N-methyl, N-trimethylsilyl trifluoroacétamide (MSTFA) and the analysis was performed by GC-MS in SIM mode. Ions monitored were: m/z 450, 482, 506 for BPR, m/z 468, 500, 524 for norBPR and m/z 455. 414, 324 for internal standard (underlined ions were used as quantifiers). Method validation was performed by: evaluation of accuracy and precision; analysis of seven drug-free hair samples; testing of linearity (0-0.5 ng/mg, n = 5). Intra-day (n = 7) and inter-day (n = 3 on 5 different days) precision were better than 8.8%for both analytes and accuracy better than 15%. The limit of detection was 0.005 ng/mg and the limit of quantitation was 0.01 ng/mg. This method was applied to hair samples collected from patients in withdrawal treatment programmes and demonstrated its good applicability in routine analysis
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